Axoplasmic vesicles that translocate on isolated microtubules in an ATP-dependent manner have an associated ATP-binding polypeptide with a previously estimated relative molecular mass of 292 kD (Gilbert, S. P., and R. D. Sloboda. 1986. J. Cell Biol. 103:947-956). Here, data are presented showing that this polypeptide (designated H1) and another high molecular mass polypeptide (H2) can be isolated in association with axoplasmic vesicles or optic lobe microtubules. The H1 and H2 polypeptides dissociate from microtubules in the presence of MgATP and can be further purified by gel filtration chromatography. The peak fraction thus obtained demonstrates MgATPase activity and promotes the translocation of salt-extracted vesicles (mean = 0.87 microns/s) and latex beads (mean = 0.92 microns/s) along isolated microtubules. The H1 polypeptide binds [alpha 32P]8-azidoATP and is thermosoluble, but the H2 polypeptide does not share these characteristics. In immunofluorescence experiments with dissociated squid axoplasm, affinity-purified H1 antibodies yield a punctate pattern that corresponds to vesicle-like particles, and these antibodies inhibit the bidirectional movement of axoplasmic vesicles. H2 is cleaved by UV irradiation in the presence of MgATP and vanadate to yield vanadate-induced peptides of 240 and 195 kD, yet H1 does not cleave under identical conditions. These experiments also demonstrate that the actual relative molecular mass of the H1 and H2 polypeptides is approximately 435 kD. On sucrose density gradients, H1 and H2 sediment at 19-20 S, and negatively stained samples reveal particles comprised of two globular heads with stems that contact each other and extend to a common base. The results demonstrate that the complex purified is a vesicle-associated ATPase whose characteristics indicate that it is a squid isoform of dynein. Furthermore, the data suggest that this vesicle-associated dynein promotes membranous organelle motility during fast axoplasmic transport.